Stat1 and NF-kappaB play important roles in the regulation of the immune system. Abnormal regulation of STAT and NF-kappaB activities has been associated with various human immune disorders. The activation of Stat1 and NF-kappaB has a central role in initiating the innate immune response. Studies from my laboratory have identified PIAS1 (protein inhibitor of activated Stat1) as a common negative regulator of Stat1 and NF-kappaB. Recently, we have generated PIAS1 null mice. Our preliminary studies have identified a physiological role of PIAS1 in the regulation of immune responses. The overall goal of this research proposal is to study the role of PIAS1 in immune regulation. These studies will enhance our ability to design novel therapeutic strategies for the treatment of infectious diseases. We will characterize the immunological phenotypes of Piasl knockout mice and examine the role of PIAS1 in innate immunity to pathogens. We will study the signaling specificity of PIAS1. Microarray analysis will be performed to define the specificity of PIAS1 in regulating the IFN-induced Stat1-dependent gene activation. We will examine the potential effect of Stat1 binding affinity on the specificity of PIAS1 using in vitro and in vivo DNA binding assays. In addition, we will test if PIASy affects the specificity of PIAS1 in Stat1-dependent gene activation by gene targeting and RNA interference approaches. We will examine the molecular mechanisms involved in the PIASl-mediated effect in immune regulation. Specifically, we will investigate the physiological importance of the PIAS1 SUMO ligase activity in PIASl-mediated gene regulation using a gene targeting strategy. The significance of the PIASl-mediated NF-kappaB regulation and the involvement of PIASl-regulated Stat1-dependent gene activation in antiviral response will be examined.